Novel prrs virus inducing type i interferon in susceptible cells

ABSTRACT

The present invention relates to the field of attenuated live viruses useful as vaccine or medicament for preventing or treating Porcine Reproductive and Respiratory Syndrome (PRRS) in swine, and is based on the surprising finding of a PRRS virus which is able to induce the interferon type I response of a cell infected by said virus. In one embodiment, the PRRS virus according to the invention is a PRRS virus mutant comprising, in comparison with the genome of a wild type strain, a mutation in the gene encoding the non structural protein 1 (nsp1) of said virus.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 1, 2014 isnamed 01-2738-SEQ-US-2.txt and is 243,257 bytes in size.

FIELD OF THE INVENTION

The present invention belongs to the field of vaccines and medicamentsfor the prophylaxis and treatment of infectious diseases. In particular,it relates to attenuated live viruses useful as vaccine or medicamentfor preventing or treating Porcine Reproductive and Respiratory Syndrome(PRRS), a viral disease affecting swine.

BACKGROUND OF THE INVENTION

Porcine reproductive and respiratory syndrome virus (PRRSV) is a memberof the virus family Arteriviridae and belongs, together with theCoronaviridae, to the virus order Nidovirales. PRRSV is an envelopedvirus with a single-stranded, positive-sense RNA genome of about 15kilobases comprising nine open reading frames (ORFs), namely ORF1a,ORF1ab, ORF2a, ORF 2ab, and ORFs 3 through ORF7. ORFs1a and 1ab encodelarge polyproteins that are processed into the viral nonstructuralproteins (nsp) by auto- and transcleavages of viral proteases nsp1,nsp2, and nsp4 (Snijder and Meulenberg, 1998).

There are two distinct viral PRRSV genotypes causing similar clinicalsymptoms that diverge by about 40% on nucleotide sequence level,genotype I (EU) and genotype II (US). The North American (US) prototypestrain is VR-2332, while the European (EU) prototype strain is Lelystadvirus.

PRRSV is considered one of the economically most important infectiousagents in pigs causing late-term reproductive failure in sows andrespiratory disease in growing pigs. Often, PRRSV infection iscomplicated by secondary bacterial infections being attributed to theimmunosuppressive nature of the virus. Also, PRRSV viremia lasts forweeks, and virus then still can be detected in lymphoid organs forseveral months, demonstrating difficulties or failure of the host'simmune response to clear the virus (Allende et al., 2000).

The specific immune response to PRRSV infection is characterized bydelayed induction of neutralizing antibodies (Lopez and Osorio, 2004)and short cell-mediated immune response (Xiao et al., 2004). It iscommonly accepted that these effects can in part be attributed, alongwith presentation of decoy epitopes (Ostrowski et al., 2002; Ansari etal., 2006) and glycan shielding of viral envelope proteins (Ansari etal., 2006), to the viral inhibition of the host's innate immune system.It has been demonstrated that PRRSV infection does not or only weakly ordelayedly induce production of type I interferon (IFN) (interferon α andinterferon β; (Miller et al., 2004)) or type II IFN, (interferon α;(Meier et al., 2003)) in susceptible cell lines (swine pulmonaryalveolar macrophages, monkey kidney cells MARC-145) and/or pigs(Buddaert et al., 1998).

Thus, there is a need for novel vaccines and medications effecting arapid induction of neutralizing antibodies and interferon responses forthe prophylaxis and treatment of PRRSV infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Sequence alignment of nsp1β protein sequences from parental EUPRRSV strain LoN94-13 and from vaccine candidates.

FIG. 2: Sequence alignment of nsp1β proteins from PRRSV strains.

FIG. 3A: PRRSV-specific immunofluorescence of virus stock titrations ofdelta nsp1 XVII-1.Black, negative fluorescence; light grey, few positivecells; grey, foci of positive cells; dark grey, complete cell monolayerpositive.

FIG. 3B: PRRSV-specific immunofluorescence of virus stock titrations ofdelta nsp1 XVIII-12. Black, negative fluorescence; light grey, fewpositive cells; grey, foci of positive cells; dark grey, complete cellmonolayer positive.

FIG. 4: Virus stocks of vaccine candidates contain IFNβ.

FIG. 5: Time course of IFNβ induction after infection of MA104 cells.

FIG. 6: Delta nsp1 mutants show increased sensitivity to type I IFN.

FIG. 7: Mean body temperatures of vaccinated groups before and afterchallenge.

DETAILED DESCRIPTION

IFNs play an important role in establishing an effective adaptive immuneresponse against viral infections, and many viruses therefore havedeveloped strategies and counteractions against onset of the host'sinnate immune system (Haller and Weber, 2009). In the interest toidentify the anticipated PRRSV IFN antagonist(s), extensive screeninganalyses based on cell lines stably expressing genes of interest or oncells transfected with protein-expressing plasmids have identifiedseveral PRRSV nonstructural proteins (nsps) including nsp1 (see below),nsp2 (Beura et al., 2010; Li et al., 2010), nsp4 (Beura et al., 2010),and nsp11 (Beura et al., 2010; Shi et al., 2011a) to be involved inblocking the induction of type I IFN.

nsp1 is located at the N-terminus of the PRRSV ORF1a-derived polyprotein1a and is processed into two multifunctional subunits, nsp1α and nsp1β,each of which contains a papain-like cystein protease (PCP) domainessential for self-release from the viral polyprotein (den Boon et al.,1995; Chen et al., 2010). nsp1α contains an N-terminal zinc-fingerdomain and the PCPα protease domain, while nsp1β contains PCPβ. For bothnsp1 subunits, nsp1α and nsp1β, the three-dimensional crystal structurehas been resolved (Sun et al., 2009; Xue et al., 2010). According tothese analyses, nsp1β consists of an N-terminal domain (NTD), a linkerdomain (LKD), the PCP domain (PCP beta), and a C-terminal extension(CTE); (Xue et al., 2010), see FIG. 2. C-terminal, nsp1β-mediatedcleavage of nsp1 from nsp2 occurs at site WYG/AGR for PRRSV US strains(Kroese et al., 2008) or is predicted at site WYG/AAG for PRRSV EUstrains (Chen et al., 2010), while nsp1α/nsp1β cleavage occurs at siteECAM/AxVYD for PRRSV US strains or is predicted at site EEAH/SxVYR forPRRSV EU strains (Chen et al., 2010).

Several studies on protein level demonstrated to the mechanistic detailthat PRRSV nsp1 and/or its autocleavage-derived subunits nsp1α and/ornsp1β inhibit type I IFN production by interfering with IFNtranscription (Song et al., 2010; Kim et al., 2010; Chen et al., 2010;Beura et al., 2010). In addition, it has been demonstrated that nsp1βinterferes with the cellular response to interferon (interferonsignaling); (Chen et al., 2010). Moreover, it was demonstrated thatPRRSV infection inhibits IFNα and/or IFNβ production in PRRSV infectedcells in vitro (Kim et al., 2010; Beura et al., 2010), the subcellularlocalization of nsp1(subunits) was determined (Song et al., 2010; Chenet al., 2010), and mechanistic aspects of type I IFN inhibition thatwere obtained by others from single protein expression experiments wereconfirmed in cells infected with PRRSV (Shi et al., 2010). Finally, ansp1 mutagenesis study based on nsp1 protein expression investigatedeffects on viral IFN inhibition (Shi et al., 2011b), showing thatmutations that inactivated papain-like cysteine protease activity ofnsp1α made nsp1 lose its IFN antagonism activity, whereas mutations thatinactivated papain-like cysteine protease activity of nsp1β did notinfluence the IFN antagonism activity of nsp1.

However, a viable PRRSV strain that induces IFN production and/or doesnot interfere with IFN signaling after infection of susceptible cellsand/or the host, in particular a PRRSV strain comprising a genomicmutation in its nsp1 gene, has not been described yet.

It is thus an aim of the present invention to make available such aviable PRRS virus inducing the IFN response of a cell, in particular ofa host cell, wherein said PRRS virus may serve as an effective vaccineor medicament for the prophylaxis or treatment of the Porcinereproductive and respiratory syndrome in swine.

The solution to the above technical problem is achieved by thedescription and the embodiments characterized in the claims.

Thus, the invention in its different aspects and embodiments isimplemented according to the claims.

The invention is based on the surprising finding of a PorcineReproductive and Respiratory Syndrome (PRRS) virus which is able toinduce the interferon type I response of a cell, in particular of a hostcell.

Hence, one aspect of the invention concerns a PRRS virus (PRRSV) whichis able to induce the interferon type I production and secretion by acell infected by said virus, wherein the PRRS virus according to theinvention in particular is able to induce or induces the interferon typeI production and secretion by an interferon competent cell infected bysaid virus.

As used herein, it is understood that the terms “interferon type I”,“IFN type I”, “type I interferon” and “type I IFN” are equivalent.

Interferon type I production and secretion by a cell, as describedherein, particularly means that interferon type I is made and releasedby a cell in response to the infection of the PRRS virus according tothe invention. In this regard, interferon type I, as mentioned herein,is preferably interferon-α and/or interferon-β.

The infection of a cell by the PRRS virus according to the invention inparticular includes attachment of the virus to the cell, entry of thevirus into the cell, disassembly of the virion, and preferablyreplication and transcription of the viral genome, expression of viralproteins, and assembly and release of new infectious viral particles.

The cell, as mentioned herein, is a primary or secondary susceptiblecell, preferably a mammalian cell, in particular a porcine or a simiancell, more preferably said cell is a porcine macrophage or a MA-104 cellor a MARC-145 cell or a Vero cell.

It is further understood that the PRRS virus according to the inventionis able to induce in vitro and/or in vivo the interferon type Iproduction and secretion by a cell infected by said virus.

Preferably, the PRRS virus according to the invention is a live PRRSvirus and/or a modified-live PRRS virus and/or an attenuated PRRS virus.

The term “attenuated PRRS virus”, as described herein, is in particulardirected to a PRRS virus which is attenuated in vitro and/or in vivo,more particular in susceptible cell lines and/or the host.

The term “host”, as used herein, is in particular directed to animalsinfectable with PRRS virus, in particular swine, more particular pigs,such as domestic pigs.

As mentioned herein, “attenuated” particularly relates to a reducedvirulence of a pathogen, in particular of a wild type PRRS virus,wherein “virulence” is understood to be the degree of pathogenicity, andwherein “pathogenicity” is directed to the ability of the pathogen toproduce clinical symptoms in the host, such as elevated bodytemperature.

In particular preferably, the PRRS virus according to the invention hasor shows increased sensitivity to type I INF when compared to wild typePRRSV, wherein the term “sensitivity to type I INF” is understood asreduced viral infectivity when IFNβ is present, preferably in asufficient amount for significantly reducing viral infectivity of wildtype PRRSV, in the medium surrounding the virus at the time ofinfection.

The term “wild type PRRS virus” or “wild type PRRSV”, respectively, asused herein, is in particular directed to an infectious pathogenic PRRSvirus, which is particularly capable of causing PRRS in swine. In oneparticular preferred embodiment, the term “wild type PRRS virus” isdirected to a PRRS virus whose genome comprises a RNA sequence orconsists of a RNA polynucleotide, wherein said RNA sequence or RNApolynucleotide is a RNA copy of a polynucleotide selected from the groupconsisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

In one embodiment, the PRRS virus according to the invention is a PRRSvirus mutant, in particular comprising, in comparison with the genome ofa wild type PRRSV strain, a mutation in a gene encoding a protein ofsaid virus.

In a preferred embodiment, the PRRS virus according to the inventioncomprises a mutation in the gene encoding the nsp1 protein of saidvirus. Thus, the invention preferably concerns a PRRS virus which isable to induce the interferon type I production and secretion by aninfected cell as a result of a mutation in the gene encoding the nsp1protein, wherein said mutation is preferably a mutation as mentionedhereinafter.

Hence, the invention particularly concerns a PRRS virus comprising, incomparison with the genome of a wild type strain, a mutation in the geneencoding the nsp1 protein. Accordingly, the mutation as described hereinis preferably a mutation in comparison with the sequence of thecorresponding wild type gene encoding the nsp1 protein.

The invention is thus preferably directed to a PRRS virus; in particulara wild type PRRS virus, wherein a mutation, preferably a mutation asmentioned hereinafter, has been implemented in the genome of said virusresulting in a non-natural nsp1 protein or in the lack of nsp1 proteinof said virus.

In the context of the invention it is understood, that the mutation asdescribed herein may be implemented to any PRRS virus, such as to a PRRSvirus selected from the group consisting of wild type PRRS virus,attenuated PRRS virus, modified-life PRRS virus, PRRS virus mutant andcombinations thereof. In one example, the mutation as described hereinmay be implemented to an attenuated and/or live PRRS virus, for exampleto a PRRS virus selected from the group of the PRRS virus strains thathave been deposited on 27 Oct. 2004 with the European Collection of CellCultures (ECACC), Porton Down, Salisbury, Wiltshire, SP4 OJG, GreatBritain, under the Accession Numbers ECACC 04102703, ECACC 04102702, andECACC 04102704. Hence, the mutation as described herein can be combinedwith one or more other mutations, preferably one ore more otherattenuating mutations, in a PRRS virus.

In the context of the invention, the term “PPRRS virus” is in particularequivalent with “PRRS virus strain”.

The term “mutation” in the context of the invention is understood as achange in a genomic sequence, in particular in the RNA sequence of aPRRS virus. Since viruses that use RNA as their genetic material haverapid mutation rates, the term “mutation”, as mentioned herein, isparticularly directed to a genetically engineered change in a genomicsequence, such as by site directed mutagenesis, which in particularresults in a virus growing to titers significantly lower than wild typePRRS virus in interferon competent cells and/or in the infected host,when propagated under the same conditions. Moreover, in anotherpreferred embodiment the mutation described herein can also be caused bynatural mutation and subsequent isolation of the PRRS virus according tothe invention, wherein said isolated virus includes the mutationdescribed herein.

Preferably, the mutation, as described herein, comprises or consists ofone or more point mutations and/or one or more genomic deletions and/orone or more insertions.

The term “nsp1 protein”, as used herein, is directed to the PRRSVnonstructural protein 1.

The nsp1 protein is preferably a polypeptide having at least 70%,preferably at least 80%, more preferably at least 90%, still morepreferably at least 95% or in particular 100% sequence identity with apolypeptide selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:2 if the PRRS virus according to the invention is a genotype I PRRSvirus, or the nsp1 protein is preferably a polypeptide having at least70%, preferably at least 80%, more preferably at least 90%, still morepreferably at least 95% or in particular 100% sequence identity with thepolypeptide set forth in SEQ ID NO:3 if the PRRS virus according to theinvention is a genotype II PRRS virus.

In one aspect, the PRRS virus according to the invention preferably alsomay comprise a mutation in the gene encoding the nsp1 protein selectedfrom the group consisting of SEQ ID NOs 1-3.

It is hence understood that the PRRS virus according to the invention isa genotype I PRRS virus or a genotype II PRRS virus. It is furtherunderstood that the terms “genotype I” and “genotype II” are equivalentto the terms “genotype 1” and “genotype 2” or to the terms “type 1” and“type 2”, as frequently used in the literature in the context of PRRSV.

Sequence identity in the context of the invention is understood as beingbased on pairwise determined similarity between nucleotide or proteinsequences. The determination of percent identity between two sequencesis preferably accomplished using a mathematical algorithm, in particularthe well-known Smith-Waterman algorithm (Smith and Waterman, M. S.(1981) J Mol Biol, 147(1):195-197). For purposes of the presentinvention, percent sequence identity of an amino acid sequence isdetermined using the Smith-Waterman homology search algorithm using anaffine 6 gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (1981) Adv. Appl. Math2:482-489, herein incorporated by reference. A variant may, for example,differ from the reference nsp1, nsp1α, nsp1β or NTD molecule by as fewas 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue. Alternatively, percent identity of a nucleotide sequence isdetermined using the Smith-Waterman homology search algorithm using agap open penalty of 25 and a gap extension penalty of 5. Such adetermination of sequence identity can be performed using, for example,the DeCypher Hardware Accelerator from TimeLogic Version G, or thesequence identity is determined with the software CLC MAIN WORKBENCH4.1.1 (CLC BIO).

The term “having 100% sequence identity”, as used herein, is understoodto be equivalent to the term “being identical”.

In another preferred embodiment, the mutation in the PRRS virusaccording to the invention comprises or consists of one or more pointmutations and/or one or more genomic deletions and/or one or moreinsertions.

In a further preferred embodiment, the PRRS virus according to theinvention comprises a mutation in the gene sequences encoding the nsp1αsubunit of the nsp1 protein and/or the nsp1β subunit of the nsp1 proteinof said virus.

The term “nsp1α”, as mentioned herein, is thus directed to the PRRSVnsp1α subunit of the nsp1 protein, and the term “nsp1β”, as used herein,is hence directed to the PRRSV nsp1β subunit of the nsp1 protein.

The nsp1α is preferably a polypeptide having at least 70%, preferably atleast 80%, more preferably at least 90%, still more preferably at least95% or in particular 100% sequence identity with a polypeptide selectedfrom the group consisting of SEQ ID NO:4 and SEQ ID NO:5 if the PRRSvirus according to the invention is a genotype I PRRS virus, or thensp1α is preferably a polypeptide having at least 70%, preferably atleast 80%, more preferably at least 90%, still more preferably at least95% or in particular 100% sequence identity with the polypeptide setforth in SEQ ID NO:6 if the PRRS virus according to the invention is agenotype II PRRS virus.

The nsp1β is preferably a polypeptide having at least 70%, preferably atleast 80%, more preferably at least 90%, still more preferably at least95% or in particular 100% sequence identity with a polypeptide selectedfrom the group consisting of SEQ ID NO:7 and SEQ ID NO:8 if the PRRSvirus according to the invention is a genotype I PRRS virus, or thensp1β is preferably a polypeptide having at least 70%, preferably atleast 80%, more preferably at least 90%, still more preferably at least95% or in particular 100% sequence identity with the polypeptide setforth in SEQ ID NO:9 if the PRRS virus according to the invention is agenotype II PRRS virus.

In a particular preferred embodiment, the PRRS virus according to theinvention comprises a mutation in the gene sequence encoding theN-terminal domain (NTD) of the nsp1β of said virus. The term “NTD”, asmentioned herein, is thus directed to the N-terminal domain of the PRRSVnsp1β subunit.

The NTD is preferably a polypeptide or has a polypeptide sequence,respectively, having at least 60%, particularly at least 70%, preferablyat least 80%, more preferably at least 90%, still more preferably atleast 95% or in particular 100% sequence identity with a polypeptide ora polypeptide sequence, respectively, selected from the group consistingof SEQ ID NO:10 and SEQ ID NO:11 if the PRRS virus according to theinvention is a genotype I PRRS virus, or the NTD is preferably apolypeptide or has polypeptide sequence, respectively, having at least60%, particularly at least 70%, preferably at least 80%, more preferablyat least 90%, still more preferably at least 95% or in particular 100%sequence identity with the polypeptide or the polypeptide sequence,respectively, set forth in SEQ ID NO:12 if the PRRS virus according tothe invention is a genotype II PRRS virus.

In a preferred embodiment, the NTD comprising the mutation has thesequence selected from the group consisting of the sequences

SXXYXXXXXVXFXDXXXXGXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXXXGXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXXXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,as set forth in SEQ ID NOs:36-41, if the PRRS virus according to theinvention is a genotype I PRRS virus, or the NTD comprising the mutationpreferably has the sequence selected from the group consisting of thesequences

AXVYDIGXXAVMXVAXXXXSWAGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXXXSWGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXXXSGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXXXGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXXGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXGGXXXXFXXXXXXLXLXAXXXXXS,as set forth in SEQ ID NOs: 42-47, if the PRRS virus according to theinvention is a genotype II PRRS virus.

Thus, the PRRS virus according to the invention preferably comprises agene sequence encoding a NTD selected from the sequences

SXXYXXXXXVXFXDXXXXGXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXXXGXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXXXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,SXXYXXXXXVXFXDXXMWXXXSXXSXXLEXLPXXLXXXXXXLXRS,as set forth in SEQ ID NOs:36-41, if the PRRS virus according to theinvention is a genotype I PRRS virus, or the PRRS virus according to theinvention preferably comprises a gene sequence encoding a NTD selectedfrom the sequences

AXVYDIGXXAVMXVAXXXXSWAGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXXXSWGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXXXSGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXXXGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXXGGXXXXFXXXXXXLXLXAXXXXXS,AXVYDIGXXAVMXVAXXGGXXXXFXXXXXXLXLXAXXXXXS,as set forth in SEQ ID NOs: 42-47, if the PRRS virus according to theinvention is a genotype II PRRS virus.

In one exemplary embodiment, the NTD comprising the mutation may, forinstance, have the sequence selected from the group consisting of thesequences

SSVYRWKKFVVFTDSSXNGRMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSSXNGMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSSXNMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSSXMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSSMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSMMWTPESDDSAXLEXLPPELERQVEILIRS,as set forth in SEQ ID NOs:48-53, if the PRRS virus according to theinvention is a genotype I PRRS virus, or the NTD comprising the mutationmay, for instance, have the sequence selected from the group consistingof the sequences

ATVYDIGXXAVMYVAXXKVSWAGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXKVSWGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXKVSGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXKVGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXKGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXGGXEVKFEXVPXELKLXANRLXTS,as set forth in SEQ ID NOs: 54-59, if the PRRS virus according to theinvention is a genotype II PRRS virus.

Thus, in one exemplary embodiment, the PRRS virus according to theinvention may, for instance, comprise a gene sequence encoding a NTDselected from the sequences

SSVYRWKKFVVFTDSSXNGRMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSSXNGMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSSXNMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSSXMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSSMMWTPESDDSAXLEXLPPELERQVEILIRS,SSVYRWKKFVVFTDSMMWTPESDDSAXLEXLPPELERQVEILIRS,as set forth in SEQ ID NOs:48-53, if the PRRS virus according to theinvention is a genotype I PRRS virus, or the PRRS virus according to theinvention may, for instance, comprise a gene sequence encoding a NTDselected from the sequences

ATVYDIGXXAVMYVAXXKVSWAGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXKVSWGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXKVSGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXKVGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXKGGXEVKFEXVPXELKLXANRLXTS,ATVYDIGXXAVMYVAXXGGXEVKFEXVPXELKLXANRLXTS,as set forth in SEQ ID NOs: 54-59, if the PRRS virus according to theinvention is a genotype II PRRS virus.

It is thus understood that a genotype I PRRS virus, as mentioned herein,is in particular a virus whose genome comprises a gene sequence codingfor a polypeptide having at least 70%, preferably at least 80%, morepreferably at least 90%, still more preferably at least 95% or inparticular 100% sequence identity with a polypeptide selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:7 and SEQ ID NO:8, or is in particular a virus whose genomecomprises a gene sequence coding for a polypeptide having at least 60%,particularly at least 70%, preferably at least 80%, more preferably atleast 90%, still more preferably at least 95% or in particular 100%sequence identity with a polypeptide selected from the group consistingof SEQ ID NO:10 and SEQ ID NO:11.

Preferably, the genotype I PRRS virus, as mentioned herein, comprises orconsists of a RNA polynucleotide having at least 70%, preferably atleast 80%, more preferably at least 90%, still more preferably at least95% or in particular 100% sequence identity with a RNA polynucleotidecomplementary to a polynucleotide selected from the group consisting ofSEQ ID NO: 13 and SEQ ID NO: 14.

It is further understood that a genotype II PRRS virus, as mentionedherein, is in particular a virus whose genome comprises a gene sequencecoding for a polypeptide having at least 70%, preferably at least 80%,more preferably at least 90%, still more preferably at least 95% or inparticular 100% sequence identity with a polypeptide selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9, or is inparticular a virus whose genome comprises a gene sequence coding for apolypeptide having at least 60%, particularly at least 70%, preferablyat least 80%, more preferably at least 90%, still more preferably atleast 95% or in particular 100% sequence identity with the polypeptideset forth in SEQ ID NO:12.

Preferably, the genotype II PRRS virus, as mentioned herein, comprisesor consists of a RNA polynucleotide having at least 70%, preferably atleast 80%, more preferably at least 90%, still more preferably at least95% or in particular 100% sequence identity with a RNA polynucleotidecomplementary to the polynucleotide set forth in SEQ ID NO: 15.

In the following, the single letter code for amino acids is used foramino acid sequences, where additionally X signifies any geneticallyencoded amino acid residue.

Within the context of the invention it is in particular understood thatthe N-terminal domain (NTD) of nsp1β starts with the N-terminus ofnsp1β, preferably starts with the amino acid sequence SXXY if the PRRSvirus is a genotype I PRRS virus or with the amino acid sequence AXVY ifthe PRRS virus is a genotype II PRRS virus, and the NTD ends with theserine residue (S) within the amino sequence SFP of the nsp1β. Regardingthe NTD domain of a genotype II PRRS virus it is in particular referredto the publication Xue et al. 2010, hereby incorporated by reference.Said amino acid sequences SXXY or AXVY and SFP are especially conservedin the nsp1β of wild type PRRS viruses and are thus preferably usedamino acid sequence motifs for defining the NTD domain according to theinvention.

In a particular preferred embodiment, the PRRS virus according to theinvention thus comprises a mutation in the gene sequence coding for theNTD, wherein the NTD preferably starts with motif SXXY for PRRS virustype 1 strains or with motif AXVY for PRRS virus type 2 strains and endswith the serine residue (S) within the conserved SFP motif of the nsp1βsubunit.

According to the invention, it is in particular preferred if saidmutation is located within the part of the gene sequence coding for theNTD, where the amino acid stretches folding into B sheet secondarystructures are encoded.

In one preferred aspect, the mutation, as described herein, thuscomprises a deletion or replacement of a nucleotide triplet coding foran amino acid residue located within the first 24 N-terminal amino acidresidues of the NTD sequence.

In particular, it is preferred if the mutation described hereincomprises of a deletion or replacement of 2 to 7 or more, preferablyconsecutive, nucleotide triplets each coding for an amino acid residuelocated within the first 24 N-terminal amino acid residues of the NTDsequence.

In another preferred aspect, the mutation, as mentioned herein,comprises the deletion or replacement of a nucleotide triplet coding foran amino acid residue with a charged, preferably positively charged,side chain, in particular coding for an arginine residue.

Exemplarily, the mutation mentioned herein preferably consists of adeletion of 2, 3, 4, 5, 6 or 7 consecutive nucleotide triplets codingfor the respective number (2, 3, 4, 5, 6 or 7) of consecutive amino acidresidues located within the first 24 N-terminal amino acid residues ofthe NTD sequence, wherein this mutation comprises the deletion of anucleotide triplet coding for an amino acid residue with a charged,preferably positively charged, side chain, in particular coding for anarginine residue.

The mutation, as described herein, preferably comprises or consists of adeletion or replacement of the nucleotide triplet coding for the firstarginine residue (R) located at least 21 amino acid residues inC-terminal direction from the N-terminal amino acid residue of the nsp1βNTD and/or the mutation, as mentioned herein, comprises a deletion orreplacement of the nucleotide triplet coding for the arginine residue(R) of the nsp1 amino acid sequence RXMW if the PRRS virus is a genotypeI PRRS virus or with the amino acid sequence RGG of said virus if thePRRS virus is a genotype II PRRS virus and/or the mutation, as mentionedherein, comprises or consists of a deletion or replacement of thenucleotide triplet coding for the arginine residue (R) of the amino acidsequence RMM or RGG of the nsp1 protein.

According to the invention, the phrase “replacement of a nucleotidetriplet” is understood as being a replacement of a nucleotide triplet ofthe wild type sequence by another nucleotide triplet coding for adifferent amino acid residue than the wild type sequence.

The phrase “deletion of nucleotide triplet” in the context of theinvention is directed to a deletion of a nucleotide triplet resulting inthe deletion of an amino acid residue in comparison with the wild typesequence.

In particular, said mutation further comprises a deletion or replacementof one nucleotide triplet coding for the amino acid residue flankingsaid arginine residue (R) in N-terminal direction, wherein said mutationpreferably comprises or consists of a deletion or replacement of twoconsecutive nucleotide triplets coding for the amino acid residues PR.

More particular, said mutation further comprises a deletion orreplacement of two, three, four, five, six, seven or more consecutivenucleotide triplets coding for two, three, four, five, six, seven ormore amino acid residues flanking said arginine residue (R) inN-terminal direction.

Preferably, said mutation comprises a deletion or replacement of threeconsecutive nucleotide triplets coding for the amino acid sequence RXRor APR of the nsp1β, or wherein said mutation comprises a deletion orreplacement of four consecutive nucleotide triplets coding for the aminoacid residues GRXR or WAPR of the nsp1β.

As result, the PRRS virus according to the invention comprises in oneembodiment a gene coding for a nsp1 protein selected from the groupconsisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 andSEQ ID NO:20, or comprises in another embodiment a gene sequence codingfor a nsp1β selected from the group consisting of SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, or comprises in afurther embodiment a gene sequence coding for a NTD selected from thegroup consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29 and SEQ ID NO:30.

In yet another preferred embodiment, the mutation, as described hereincomprises a deletion of the gene sequence encoding the (whole) nsp1protein, encoding the (whole) nsp1α subunit, encoding the (whole) nsp1βsubunit, or encoding a part, preferably at least seven consecutive aminoacid residues, of or the (whole) NTD domain of the nsp1β subunit.

Yet another aspect of the invention is directed to a polynucleotidecomprising or consisting of the genome of the PPRS virus according tothe invention.

Further, the invention is also directed to a virus particle, whereinsaid virus particle comprises a polynucleotide which comprises orconsists of the genome of the PPRS virus according to the invention orwhich comprises or consists of a DNA copy of the PPRS virus according tothe invention.

Still further, the present invention provides a DNA-Vector comprising acopy of, or a cDNA sequence complementary to, respectively, apolynucleotide which comprises or consists of the genome of the PPRSvirus according to the invention.

In one exemplary and non-limiting example the DNA vector, as mentionedherein, comprises a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 andSEQ ID NO:35.

Also, the present invention provides a cell comprising a polynucleotide,which comprises or consists of the genome of the PPRS virus according tothe invention or which comprises or consists of a DNA copy of the PPRSvirus according to the invention.

In one exemplary and non-limiting example the PRRS virus according tothe invention, or the genome of the PRRS virus according to theinvention, respectively, comprises a RNA sequence or consists of a RNApolynucleotide, wherein said RNA sequence or RNA polynucleotide is a RNAcopy of a polynucleotide selected from the group consisting of SEQ IDNO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.

Another aspect of the invention concerns the PRRS virus according to theinvention for use as vaccine or medicament, in particular for use in theprophylaxis or treatment of Porcine Reproductive and RespiratorySyndrome preferably in swine. The present invention further provides acomposition containing the PRRS virus according to the invention for useas a vaccine or as a medicament, in particular for use in theprophylaxis or treatment of Porcine Reproductive and RespiratorySyndrome preferably in swine.

In a preferred aspect, said vaccine or medicament is administered in twodoses or preferably in one dose to an animal in need thereof. By theterm “prophylaxis or therapy”, as mentioned herein, it is meant that theprophylaxis is to administer a drug, in particular a vaccine, beforeexposure to PRRSV and the treatment is to administer a drug, inparticular a medicament, after infection with PRRSV or onset of PRRS.

In particular, the vaccine, as mentioned herein, is a live vaccineand/or a modified live vaccine and/or an attenuated live or attenuatedmodified live vaccine.

The strains of the PRRS virus according to the invention can be grownand harvested by methods known in the art, e.g. by propagating insuitable cells like the simian cell line MA-104, Vero cells, or porcinealveolar macrophages. Preferably, vaccines according to the presentinvention are modified live vaccines comprising one or more of thesestrains alive in a suitable carrier, but inactivated virus may also beused to prepare killed vaccine (KV). Modified live vaccines (MLV) aretypically formulated to allow administration of 10¹ to 10⁷ viralparticles per dose, preferably 10³ to 10⁶ particles per dose, morepreferably 10⁴ to 10⁶ particles per dose (4.0-6.0 log₁₀ TCID₅₀). KV maybe formulated based on a pre-inactivation titre of 10³ to 10¹⁰ viralparticles per dose. The vaccine may comprise a pharmaceuticallyacceptable carrier, for example a physiological salt-solution. Thevaccine may or may not comprise an adjuvant. A suitable adjuvant mayoptionally also be used. For example, a suitable adjuvant that may beused a-tocopherol acetate which may be obtained under the trade nameDILUVAC FORTE®. Alternatively, alum based adjuvants may be used.

A further aspect of the invention is thus directed to the use of thePRRS virus according to the invention for the preparation of a vaccineor medicament for the prophylaxis or treatment of Porcine Reproductiveand Respiratory Syndrome, preferably in swine.

A vaccine according to the present invention may be presented in form ofa freeze-dried preparation of the live virus, to be reconstituted with asolvent, to result in a solution for injection. The solvent may, forexample, be water, physiological saline, a buffer, or an adjuvantingsolvent. The solvent may additionally use adjuvants, for examplea-tocopherol acetate. The reconstituted vaccine may then be injectedinto a pig, for example as an intramuscular or intradermal injectioninto the neck. For intramuscular injection, a volume of 2 ml may beapplied, for an intradermal injection it is typically 0.2 ml. In afurther aspect, the present invention therefore is a product, comprisingin separate containers a freeze-dried composition of the virus, and asolvent for reconstitution, and optionally further containing a leafletor label comprising instructions of use.

Yet another aspect of the invention thus concerns a method for theprophylaxis or treatment of Porcine Reproductive and RespiratorySyndrome comprising administering the PRRS virus according to theinvention to an animal, preferably to a swine, more preferably to a pig,in particular preferably to a piglet or a sow.

A vaccine according to the present invention may not only comprise thePRRS virus according to the invention as antigen, but may includefurther components active against PRRS or other porcine viral orbacterial diseases, like porcine circovirus, classical swine fever virusor mycoplasma hyorhinis. Therefore, the invention further relates to avaccine as described, characterized in that it contains at least onefurther antigen active against a porcine disease which is not PRRS.

Still another aspect of the invention thus concerns a medicament orvaccine comprising the PRRS virus according to the invention.

Still a further aspect of the invention is directed to a method ofpreparing a vaccine or medicament, in particular for the prophylaxis ortreatment of Porcine Reproductive and Respiratory Syndrome preferably inswine, said method comprising the cultivation of the PRRS virusaccording to the invention in cell culture.

Moreover, the invention comprises a method of producing the PRRS virusaccording to the invention, said method comprising the mutagenesis ofthe gene coding for the nsp1 protein in the genome of a PRRS virus, andwherein the mutation, as described herein, is in particular introducedby genetic engineering in the genome of said virus, preferably by sitedirected mutagenesis.

Preferably, for any of the aforementioned methods, the above-mentionedDNA-vector is used and/or said methods comprise the detection ofinterferon type I, in particular interferon type I secretion, and/or ofmRNA coding for interferon type I. Said detection in particularcomprises the detection of interferon type I and/or of mRNA coding forinterferon type I by a bioassay, wherein said bioassay is preferablyselected from the group consisting of ELISA, PCR, GLISA, IFA,biosensoric measurement, Surface Plasmon Resonance (SPR) measurement,selective media, lateral flow, biochip measurement, immunomagneticseparation, electrochemiluminescence, chromogenic media,immunodiffusion, DNA hybridization, staining, colorimetric detection,luminescence, and combinations thereof.

In yet a further aspect, the invention provides an immunogeniccomposition containing the PRRS virus according to the invention.

As used herein, the term “immunogenic composition” in particular refersto a composition that will elicit an immune response in an animal thathas been exposed to the composition. An immune response may includeinduction of antibodies and/or induction of a T-cell response.

Usually, an “immune response” includes but is not limited to one or moreof the following effects: the production or activation of antibodies, Bcells, helper T cells, suppressor T cells, and/or cytotoxic T cells,directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. Preferably, the host will displayeither a therapeutic or a protective immunological (memory) responsesuch that resistance to new infection will be enhanced and/or theclinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction in number or severity of, or lack ofone or more of the symptoms associated with the infection of thepathogen, in the delay of onset of viremia, in a reduced viralpersistence, in a reduction of the overall viral load and/or in areduction of viral excretion. Thus, an “immune response” in particularmeans but is not limited to the development in a subset of a cellularand/or antibody-mediated immune response to the composition or vaccineof interest.

In one aspect, the immunogenic composition of the invention ispreferably a vaccine or medicament.

As used herein, the term “viremia” is particularly understood as acondition in which PRRS virus particles circulate in the bloodstream ofan animal.

The term “animal”, as mentioned herein, is in particular directed toswine, more particular to a pig, preferably a domestic pig.

In a preferred aspect, the immunogenic composition of the inventioncomprises an amount of 10¹ to 10⁷ viral particles of the PRRS virusaccording to the invention per dose, preferably 10³ to 10⁶ particles perdose, more preferably 10⁴ to 10⁶ particles per dose.

In another preferred aspect, the immunogenic composition of theinvention comprises an amount of the PRRS virus according to theinvention which is equivalent to a virus titre of at least about 10³TCID₅₀/mL per dose, preferably between 10³ to 10⁵ TCID₅₀/mL per dose

In yet another preferred aspect, the immunogenic composition of theinvention further contains one or more pharmaceutically acceptablecarriers or excipients. Said one or more pharmaceutically acceptablecarriers or excipients are preferably selected from the group consistingof solvents, dispersion media, adjuvants, stabilizing agents, diluents,preservatives, antibacterial and antifungal agents, isotonic agents, andadsorption delaying agents.

The present invention is further directed to the immunogenic compositionof the invention for use in a method for

-   -   inducing an immune response against PRRSV, or    -   the prevention or reduction of PRRS, or    -   the prevention or reduction of PRRSV viremia and/or    -   preventing or reducing clinical symptoms, in particular the        elevated body temperature, associated with PRRSV infection, or    -   the prevention or reduction of the elevated body temperature        associated with the administration of an attenuated PRRSV        vaccine to an animal.

As used herein, the term “inducing an immune response” to an antigen orcomposition is the development of a humoral and/or cellular immuneresponse in an animal to an antigen present in the composition ofinterest.

The term “prevention” or “reduction” or “preventing” or “reducing”,respectively, as used herein, means, but is not limited to a processwhich includes the administration of a PRRSV antigen, namely of thePRRSV according to the invention which is included in the composition ofthe invention, to an animal, wherein said PRRSV antigen, whenadministered to said animal elicits or is able to elicit an immuneresponse in said animal against PRRSV. Altogether, such treatmentresults in reduction of the clinical symptoms of PRRS or of symptomsassociated with PRRSV infection, respectively. More specifically, theterm “prevention” or “preventing, as used herein, means generally aprocess of prophylaxis in which an animal is exposed to the immunogeniccomposition of the present invention prior to the induction or onset ofthe disease process (PRRS).

Herein, “reduction of PRRS” or “reduction of clinical symptomsassociated with PRRSV infection” means, but is not limited to, reducingthe number of infected subjects in a group, reducing or eliminating thenumber of subjects exhibiting clinical symptoms of infection, orreducing the severity of any clinical symptoms that are present in thesubjects, in comparison to wild-type infection. For example, it shouldrefer to any reduction of pathogen load, pathogen shedding, reduction inpathogen transmission, or reduction of any clinical sign symptomatic ofPRRSV infection, in particular of elevated body temperature. Preferablythese clinical symptoms are reduced in subjects receiving thecomposition of the present invention by at least 10% in comparison tosubjects not receiving the composition and may become infected. Morepreferably, clinical symptoms are reduced in subjects receiving thecomposition of the present invention by at least 20%, preferably by atleast 30%, more preferably by at least 40%, and even more preferably byat least 50%.

Also, the elevated body temperature usually associated with theadministration of an attenuated PRRSV vaccine to an animal is reduced insubjects receiving the composition of the present invention by at least10% in comparison to subjects receiving a conventional attenuated PRRSVvaccine. More preferably, the elevated body temperature usuallyassociated with the administration of an attenuated PRRSV vaccine isreduced in subjects receiving the composition of the present inventionby at least 20%, preferably by at least 30%, more preferably by at least40%, and even more preferably by at least 50%.

Hence, a further advantage of the PRRS virus according to the inventionis in particular the effect that its administration to an animal, e.g.for the prophylaxis or treatment of PRRS, results in a reduced increaseof body temperature in the animal in comparison with a conventionalattenuated PPRSV vaccine.

The term “conventional attenuated PRRSV vaccine”, as mentioned herein,is in particular directed to any attenuated PRRSV useful as a vaccine,wherein said PRRSV does not have the characteristic features of the PRRSvirus of the present invention, in particular is not able to induce theinterferon type I production and secretion by a cell infected by saidPRRSV.

The term “subject”, as mentioned herein, in particular relates to ananimal.

The term “body temperature”, as used herein, in particular refers to theapproximate average normal, internal temperature of an animal, forexample about 38.5-39° C. in pigs, whereas the body temperatureassociated with a PRRSV infection may be elevated up to 41° C. in pigs.

The term “reduction of PRRSV viremia” means, but is not limited to, thereduction of PRRS virus entering the bloodstream of an animal, whereinthe viremia level, i.e. the number of PRRSV RNA copies per mL of bloodserum or the number of plaque forming colonies per deciliter of bloodserum, is reduced in the blood serum of subjects receiving thecomposition of the present invention by at least 50% in comparison tosubjects not receiving the composition and may become infected. Morepreferably the viremia level is reduced in subjects receiving thecomposition of the present invention by at least 90%, preferably by atleast 99.9%, more preferably by at least 99.99%, and even morepreferably by at least 99.999%.

The present invention further relates to the use of the PRRS virusaccording to the invention or of the immunogenic composition of theinvention for the preparation of a vaccine or medicament for

-   -   inducing an immune response against PRRSV, or    -   treating or preventing PRRS, or    -   preventing or reducing PRRSV viremia and/or    -   preventing or reducing clinical symptoms, in particular the        elevated body temperature, associated with PRRSV infection, or    -   the prevention or reduction of the elevated body temperature        associated with the administration of an attenuated PRRSV        vaccine to an animal.

In particular, it is preferred if the vaccine or medicament, asmentioned herein, is to be administered in two doses or preferably in asingle dose to an animal.

The present invention also provides a method of preparing an immunogeniccomposition, preferably a vaccine or medicament, more preferably avaccine or medicament for

-   -   inducing an immune response against PRRSV, or    -   treating or preventing PRRS, or    -   preventing or reducing PRRSV viremia and/or    -   preventing or reducing clinical symptoms, in particular the        elevated body temperature, associated with PRRSV infection, or    -   the prevention or reduction of the elevated body temperature        associated with the administration of an attenuated PRRSV        vaccine to an animal,        wherein said method comprises the step of mixing the PRRS virus        according to the invention with one or more pharmaceutically        acceptable carriers or excipients, and wherein said one or more        pharmaceutically acceptable carriers or excipients are        preferably selected from the group consisting of solvents,        dispersion media, adjuvants, stabilizing agents, diluents,        preservatives, antibacterial and antifungal agents, isotonic        agents, and adsorption delaying agents.

In another aspect, the present invention further provides a method for

-   -   inducing an immune response against PRRSV, or    -   treating or preventing PRRS, or    -   preventing or reducing PRRSV viremia and/or    -   preventing or reducing clinical symptoms, in particular the        elevated body temperature, associated with PRRSV infection, or    -   the prevention or reduction of the elevated body temperature        associated with the administration of an attenuated PRRSV        vaccine to an animal,        wherein said method comprises the step of administering the        immunogenic composition of the invention to an animal in need        thereof, and wherein preferably the immunogenic composition        and/or the vaccine is administered in two doses or, more        preferably, in a single dose.

Examples

In the examples five viable, genetically designed PRRSV mutant strainsare described which are based on infectious EU PRRSV cDNA cloneLoN94-13. These strains, delta nsp1 IX-10, delta nsp1 XVII-1, delta nsp1XVIII-12, delta nsp1 XIX-2 and delta nsp1 XX-9 (henceforth referred toas vaccine candidates), harbor genomic deletions of two, three, four,five, or six codons in their predicted nsp1 genes, respectively,resulting in deletions of two (motif P21R22), three (motif R20P21R22),four (motif G19R20P21R22), five (motif N18G19R20P21R22), or six (motif(P17N18G19R20P21R22) amino acids in their predicted nsp1β proteins,respectively (FIG. 1).

Based on sequence alignments of parental strain LoN94-13 with PRRSV USand EU reference strains VR-2332 and Lelystad virus as well as withstrain GD-XH, the deletions are located in the predicted nsp1β portionof nsp1 (FIG. 2). In more detail, the deletion site for all vaccinecandidates is located in the N-terminal domain (NTD) of nsp1β andoverlaps with amino acids P23R24 of GD-XH nsp1β; (FIG. 2).

After transfection of synthetic transcripts of the vaccine candidatesinto BHK21 cells and transfer of cell culture supernatant fromtransfected BHK21 cells onto PRRSV-susceptible MA104 cells, plaqueformation typical for PRRSV infection occurred. PRRSV-specificity andviability for each of the vaccine strains then was demonstrated bysubsequent cell culture passages on MA104 cells and PRRSV-specificimmunofluorescence using monoclonal antibody SDOW17 (RuralTechnologies).

After endpoint dilution and generation of virus stocks each derived frommaterial of a single virus plaque, virus titers of the obtained virusstocks were determined for each vaccine candidate by serial virustitrations on 96-well plates containing MA104 cells followed byPRRSV-specific immunofluorescence analyses six to seven days postinfection. Unlike experience with titrations of virus stocks fromparental PRRSV LoN94-13, the first serial dilutions of vaccinecandidates delta nsp1 XVII-1 and delta nsp1 XVIII-12 did not demonstratea cytopathic effect and virus plaque formation, while at higherdilutions of the virus stocks a cytopathic effect was detectable.Moreover, when respective titrations were investigated byimmunofluorescence, cell culture wells of the first serial dilutionswere negative for PRRSV infection for both vaccine candidates, whilewells infected with higher dilutions of the virus stocks showedPRRSV-specific immunofluorescence, respectively (FIG. 3A and FIG. 3B).

To determine whether the prepared vaccine candidate virus stockscontained type I IFN, a commercial ELISA specific for human IFNβ(Invitrogen) was used. MA104 cells are epithelial Green Monkey kidneycells. According to the ELISA manufacturer, this Invitrogen ELISA isalso suited for the detection of primate IFNβ other than human. For eachvaccine candidate's virus stock, 100 μl served as assay input, while avirus stock from parental strain LoN94-13, cell culture medium, andmedium from noninfected cells served as controls. For quantification ofthe obtained results, a calibration curve was included using a positivecontrol of the ELISA manufacturer. All samples were measured induplicates. Unlike the negative controls, virus stocks of the vaccinecandidates contained considerable levels of type I IFN, while the virusstock of the parental virus showed IFN levels as low as the negativecontrols (FIG. 4).

To confirm the results obtained and to assess kinetics of type I IFNproduction in cells infected with the vaccine candidates, a time courseexperiment was performed using MA104 cells infected at a multiplicity ofinfection (MOI) of 0.001, respectively. Parental strain LoN94-13 servedas negative control. While there were only very little and unalteredlevels of type I IFN near background detectable for infection withparental strain LoN94-13, vaccine candidates delta nsp1 XVII-1 and deltansp1 XVIII-12 induced considerable and increasing amounts of up to about18 I.U. IFNβ per 25 μl sample volume from two days post infection on(FIG. 5).

It was experimentally assessed whether vaccine candidates containinggenomic deletions in nsp1 demonstrate an increased sensitivity to type IIFN (FIG. 6). 5×10⁵ MA104 cells were seeded into a well of a six-wellplate and were either not infected (n.inf.) or infected with 800infectious virus particles of one of the virus strains given on top,respectively. Cells then were either inoculated with 120 I.U. human IFNβ(+IFNβ, bottom row), respectively, or not (−IFNβ, top row). Three dayspost infection, immunofluorescence analysis specific for the PRRSVcapsid protein was performed using monoclonal antibody SDOW17 (RuralTechnologies). The total numbers of foci of PRRSV-infected cells perwell are given below, respectively.

This experiment demonstrated that inoculation with type I IFN reducedthe number of PRRSV infection events in cells after inoculation with adefined number of infectious virus particles, reflecting reduced viralinfectivity of PRRSV when IFN was added. This reduction was 80-fold forwild type virus LoN94-13 (FIG. 6). In addition, for infection with wildtype virus, foci of infected cells were smaller than in the well notinoculated with IFN (FIG. 6). However, for vaccine strains delta nsp1XVII-1 and delta nsp1 XVIII-12, viral infectivity was reduced to zerowhen INF was added (FIG. 6). Thus, these vaccine candidates not onlyinduce production of type I IFN in infected cells (FIG. 4 and FIG. 5),but also demonstrate increased sensitivity to type I INF when comparedto wild type PRRSV (FIG. 6). This is reflected by their dramaticallyreduced viral infectivity when IFNβ is present.

Interestingly, the cells infected for the time course experimentsummarized in FIG. 5 not only produced considerable amounts of IFNβ, butat the end of the experiment at six days post infection, cells infectedwith vaccine candidates delta nsp1 XVII-1 and delta nsp1 XVIII-12 showedsigns of recovery from usually lytical PRRSV infection. While cellsinfected with parental strain LoN94-13 were fully lysed, cells infectedwith the vaccine candidates grew in a partially (delta nsp1 XVIII-12) orcompletely intact monolayer (delta nsp1 XVII-1). For the latter, onlyweak signs of a PRRSV-induced cytopathic effect were still detectable.Thus, the interferon production of infected cells together with theobserved sensitivity of vaccine candidates to type I IFN correlated withpartial or almost complete recovery of infected cells over time. It isreasonable to expect that type I IFN induction by the vaccine candidatestogether with their increased sensitivity to type I IFN will contributeto a significantly attenuated viral phenotype in the natural host. Inparticular, expected features of the vaccine candidates' attenuation inpigs include stimulation of the innate and specific immunity, bothhumoral and cellular, and less shedding and/or shortened viremia of thevaccine viruses.

To assess whether the PRRSV vaccine candidates are attenuated in thehost, an animal experiment in piglets was performed.

Three groups, each of ten animals, were infected at study day 0 eitherwith wild-type parental EU PRRSV strain Lon94-13 (WT group), or withdelta nsp1 XVIII-12 (nsp1 group), or were not infected (Ch controlgroup). Infection was applied by intramuscular injection to the neck atdosages of 10^(6,56) TCID₅₀ for LoN94-13 or 10^(6,6) TCID₅₀ for deltansp1 XVIII-12, respectively. 21 days post vaccination, all animals werechallenged with a virulent EU PRRSV strain being heterologous toLoN94-13 by intramuscular injection and intranasal inoculation at atotal dosage of 3×10^(6,52) TCID₅₀. Animals were kept until the end ofthe experiment at day 31, ten days after challenge, and bodytemperatures were measured for all animals at days 0 (1 and 4 hours postvaccination), 1, 3, 5, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, and 31.

Mean body temperatures were determined for each animal for the timeafter vaccination but before challenge using measured body temperaturedata from all timepoints from day 0 through day 20. Subsequently, meanbody temperatures were determined for each group (FIG. 7, (left-hand)columns). Error bars indicate standard deviations, respectively.Following the same procedure, mean body temperatures and standarddeviations were determined for all groups for the time after challengeusing measured body temperature data from all timepoints from day 22through 31 (FIG. 7, (right-hand) columns).

The term “Significant(ly)” in the context of the following means either(i) p-values of 0.05 or lower as determined by the Dunnett test andobtained from comparing the nsp1 group with either the WT or the Chcontrol group for either of the two time periods investigated (beforeand after challenge) or (ii) p-values of 0.05 or lower when comparingthe mean temperature change within a group and between the two timeperiods investigated (before and after challenge).

When comparing the determined mean body temperatures for the time aftervaccination but before challenge (left hand columns) in between thethree groups, animals from the WT group demonstrated a rise in bodytemperature of more than 0.4° C. when compared to animals from thenoninfected Ch control group, thus demonstrating virulence of LoN94-13in the infected host. In contrast, the nsp1 group showed a significantreduction in mean body temperature of more than 0.2° C. when compared tothe WT group. Thus, since vaccination dosages were the same for the WTand the nsp1 group, the considerable reduction in increase of bodytemperature compared to WT demonstrates that the described mutation inthe genome of delta nsp1 XVIII-12 has significantly reduced virulence ofthe WT parental strain LoN94-13 in the infected animal.

The significant rise in the mean body temperature of the Ch controlgroup from before challenge to after challenge of 0.4° C. demonstratesvirulence of the heterologous EU PRRSV challenge strain. The meantemperature of the WT group after challenge was slightly lower thanbefore challenge, but not significantly reduced (FIG. 7). In contrast,the mean body temperature of the nsp1 group after challenge wassignificantly reduced by almost 0.2° C. when compared to that beforechallenge. Moreover, the body temperature of the nsp1 group afterchallenge was significantly reduced by almost 0.4° C. when compared tothe Ch control group after challenge. Also, mean body temperature of thensp1 group after challenge was significantly lower that that of the WTgroup after challenge by more than 0.2° C. Taken together, thisdemonstrates that a measurable and significant degree of protection fromsigns of disease induced by the applied challenge virus was conferred topigs by vaccination with delta nsp1 XVIII-12. Since parental PRRSVstrain LoN94-13 did not confer significant protection, it is evidentthat the described mutation in the genome of delta nsp1 XVIII-12 iscausative for the observed significant protective technical effect.

Analogous experiments, wherein the vaccination was performed with loweramounts (10⁵ TCID₅₀) of delta nsp1 XVIII-12 showed results similar tothe above described results (data not shown). Thus, in practice, apreferred amount of 10³ to 10⁵ TCID₅₀ is sufficient for vaccination.

Taken together, the invention described here represents the first knownviable PRRSV (EU) strains that contain mutations (deletions) in the nsp1gene (nsp1β) that induce type I IFN (IFNβ) production in susceptiblecells (MA104) and that show increased sensitivity to type I IFN (IFNβ).Moreover, the animal data demonstrates that (i) vaccine candidate deltansp1 XVIII-12 is significantly attenuated in the host when compared toits parental PRRSV strain LoN94-13 and that (ii) vaccine candidate deltansp1 XVIII-12 confers significant protection from signs of diseaseinduced by challenge with a heterologous PRRSV strain while parentalstrain LoN94-13 does not. Thus, the described vaccine candidates or thedescribed mutations therein, either alone or combined with otherattenuating mutations, may serve as promising life attenuated PRRSVvaccines.

In the Sequence Listing

SEQ ID NO: 1 corresponds to LoN94-13 complete nsp1 protein,

SEQ ID NO: 2 corresponds to Lelystad virus complete nsp1 protein,

SEQ ID NO: 3 corresponds to VR2332 complete nsp1 protein,

SEQ ID NO: 4 corresponds to LoN94-13 complete nsp1 Alpha,

SEQ ID NO: 5 corresponds to Lelystad virus complete nsp1 Alpha,

SEQ ID NO: 6 corresponds to VR2332 complete nsp1 Alpha,

SEQ ID NO: 7 corresponds to LoN94-13 complete nsp1 Beta,

SEQ ID NO: 8 corresponds to Lelystad virus complete nsp1 Beta,

SEQ ID NO: 9 corresponds to VR2332 complete nsp1 Beta,

SEQ ID NO: 10 corresponds to LoN94-13 nsp1Beta NTD,

SEQ ID NO: 11 corresponds to Lelystad virus nsp1 Beta NTD,

SEQ ID NO: 12 corresponds to VR2332 nsp1 Beta NTD,

SEQ ID NO: 13 corresponds to LoN94-13 complete viral cDNA insert,

SEQ ID NO: 14 corresponds to Lelystad virus complete genome,

SEQ ID NO: 15 corresponds to VR2332 complete genome,

SEQ ID NO: 16 corresponds to delta nsp1 IX-10 complete nsp1 proteinsequence,

SEQ ID NO: 17 corresponds to delta nsp1 XVII-1 complete nsp1 proteinsequence,

SEQ ID NO: 18 corresponds to delta nsp1 XVIII-12 complete nsp1 proteinsequence,

SEQ ID NO: 19 corresponds to delta nsp1 XIX-2 complete nsp1 proteinsequence,

SEQ ID NO: 20 corresponds to delta nsp1 XX-9 complete nsp1 proteinsequence,

SEQ ID NO: 21 corresponds to delta nsp1 IX-10 complete nsp1Beta proteinsequence,

SEQ ID NO: 22 corresponds to delta nsp1 XVII-1 complete nsp1Beta proteinsequence,

SEQ ID NO: 23 corresponds to delta nsp1 XVIII-12 complete nsp1 Betaprotein sequence,

SEQ ID NO: 24 corresponds to delta nsp1 XIX-2 complete nsp1 Beta proteinsequence,

SEQ ID NO: 25 corresponds to delta nsp1 XX-9 complete nsp1Beta proteinsequence,

SEQ ID NO: 26 corresponds to delta nsp1 IX-10 nsp1 Beta NTD proteinsequence,

SEQ ID NO: 27 corresponds to delta nsp1 XVII-1 nsp1Beta NTD proteinsequence,

SEQ ID NO: 28 corresponds to delta nsp1 XVIII-12 nsp1Beta NTD proteinsequence,

SEQ ID NO: 29 corresponds to delta nsp1 XIX-2 nsp1 Beta NTD proteinsequence,

SEQ ID NO: 30 corresponds to delta nsp1 XX-9 nsp1Beta NTD proteinsequence,

SEQ ID NO: 31 corresponds to delta nsp1 IX-10 complete viral cDNA insertsequence,

SEQ ID NO: 32 corresponds to delta nsp1 XVII-1 complete viral cDNAinsert sequence,

SEQ ID NO: 33 corresponds to delta nsp1 XVIII-12 complete viral cDNAinsert sequence,

SEQ ID NO: 34 corresponds to delta nsp1 XIX-2 complete viral cDNA insertsequence,

SEQ ID NO: 35 corresponds to delta nsp1 XX-9 complete viral cDNA insertsequence,

SEQ ID NO: 36 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 2 aa),

SEQ ID NO: 37 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 3 aa),

SEQ ID NO: 38 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 4 aa),

SEQ ID NO: 39 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 5 aa),

SEQ ID NO: 40 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 6 aa),

SEQ ID NO: 41 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 7 aa),

SEQ ID NO: 42 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 2 aa),

SEQ ID NO: 43 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 3 aa),

SEQ ID NO: 44 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 4 aa),

SEQ ID NO: 45 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 5 aa),

SEQ ID NO: 46 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 6 aa),

SEQ ID NO: 47 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 7 aa),

SEQ ID NO: 48 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 2 aa),

SEQ ID NO: 49 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 3 aa),

SEQ ID NO: 50 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 4 aa),

SEQ ID NO: 51 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 5 aa),

SEQ ID NO: 52 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 6 aa),

SEQ ID NO: 53 corresponds to a (type 1) nsp1 Beta NTD with mutation(deletion of 7 aa),

SEQ ID NO: 54 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 2 aa),

SEQ ID NO: 55 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 3 aa),

SEQ ID NO: 56 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 4 aa),

SEQ ID NO: 57 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 5 aa),

SEQ ID NO: 58 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 6 aa),

SEQ ID NO: 59 corresponds to a (type 2) nsp1 Beta NTD with mutation(deletion of 7 aa).

REFERENCE LIST

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1. A replication-competent Porcine Reproductive and Respiratory Syndrome (PRRS) virus comprising a deletion of at least three consecutive nucleotide triplets in the gene sequence encoding the N-terminal domain (NTD) of the nsp1β subunit of the nsp1 protein of said virus, wherein the NTD starts with the N-terminal amino acid sequence SXXY (SEQ ID NO: 60) of nsp1β and ends with the serine (S) residue within the amino acid sequence SFP (SEQ ID NO: 62), wherein said deletion of at least three consecutive nucleotide triplets comprises a deletion of the nucleotide triplet encoding the first arginine residue (R) located at least 21 amino acid residues in the C-terminal direction from the N-terminal amino acid residue of nsp1β and a deletion of the two nucleotide triplets coding for the two amino acid residues flanking said R residue in the N-terminal direction, and wherein the modified PRRS virus induces production and secretion of interferon type I by a cell infected with said virus.
 2. The PRRS virus according to claim 1, wherein said mutation comprises a deletion of three consecutive nucleotide triplets coding for the amino acid sequence RXR (SEQ ID NO: 66) of the nsp1β subunit, or wherein said mutation comprises a deletion of four consecutive nucleotide triplets coding for the amino acid residues GRXR (SEQ ID NO: 68) of the nsp1β subunit, wherein X is a genetically encoded amino acid residue.
 3. An immunogenic composition comprising the PRRS virus according to claim
 1. 4. The immunogenic composition according to claim 3, comprising an amount of 10¹ to 10⁷ viral particles per dose
 5. The immunogenic composition according to claim 3, comprising an amount of 10³ to 10⁶ particles per dose.
 6. The immunogenic composition according to claim 3, comprising an amount of 10⁴ to 10⁶ particles per dose.
 7. The immunogenic composition according to claim 3, comprising an amount of said PRRS virus which is equivalent to a virus titer of at least about 10³ TCID50/mL per dose.
 8. The immunogenic composition according to claim 3, comprising an amount of said PRRS virus which is equivalent to a virus titer of between 10³ to 10⁵ TCID50/mL per dose.
 9. The immunogenic composition according to claim 3, further comprising one or more pharmaceutically acceptable carriers or excipients.
 10. The immunogenic composition according to claim 9, wherein said one or more pharmaceutically acceptable carriers or excipients are selected from the group consisting of solvents, dispersion media, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, and adsorption delaying agents.
 11. The PRRS virus according to claim 1, wherein said PPRS virus has increased sensitivity to interferon type I.
 12. The PRRS virus according to claim 1, wherein said PRRS virus is an attenuated PRRS virus.
 13. The PRRS virus according to claim 1, wherein said PPRS virus is a genotype I virus.
 14. The PRRS virus according to claim 1, wherein said interferon type I is Interferon-α, and/or Interferon-β or combinations thereof.
 15. The PRRS virus according to claim 1, wherein said cell is a mammalian cell, and wherein said mammalian cell is a cell of a primary or secondary cell line or a host cell.
 16. The PRRS virus according to claim 15, wherein said mammalian cell is a porcine cell or a simian cell.
 17. The PRRS virus according to claim 16, wherein said porcine cell is a porcine macrophage.
 18. The PRRS virus according to claim 16, wherein said simian cell is a MA-104 or a MARC-145 cell.
 19. A polynucleotide comprising the genome of the PRRS virus according to claim
 1. 20. A DNA Vector comprising a copy of the polynucleotide of claim
 19. 21. A virus particle comprising the polynucleotide of claim
 19. 22. A cell comprising the polynucleotide of claim
 19. 23. A cell comprising the DNA-vector of claim
 20. 24. A method of preparing a PRRS virus composition comprising the cultivation of the PRRS virus according to claim 1 in cell culture.
 25. A method for the treatment of Porcine Reproductive and Respiratory Syndrome comprising administering an effective amount of the PRRS virus according to claim 1 to a swine, pig, piglet or a sow in need of said treatment.
 26. A method for inducing an immune response against PRRSV comprising administering an effective amount of the immunogenic composition according to claim 1 to a swine, pig, piglet or a sow in need thereof.
 27. A method for the detection of interferon type I and/or the mRNA coding for interferon type I, by the cell infected with the modified PRRS virus according to claim 1, wherein the interferon type I and/or the mRNA coding for the interferon type I is detected by a bioassay, wherein said bioassay is preferably selected from the group consisting of ELISA, PCR, GLISA, IFA, biosensoric measurement, Surface Plasmon Resonance (SPR) measurement, selective media, lateral flow, biochip measurement, immunomagnetic separation, electrochemiluminescence, chromogenic media, immunodiffusion, DNA hybridization, staining, colorimetric detection, luminescence, and combinations thereof.
 28. The method according to claim 24, wherein the immunogenic composition is administered in a single dose or in two doses. 